FIG. 9.

Effects of insulin and calyculin A on Ser phosphorylation of PH/PTB-L mutants. (A) CHO-T cells were transiently transfected with constructs expressing either Myc-PH/PTB^WT or the following PH/PTB mutants: S336A, S407A, S408A, S407A/S408A (S407/8A), and S336A/S407A/S408A (S3A). After 24 h, the cells were deprived of serum for 16 h and treated with insulin for the indicated times. Cell extracts were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-Myc (α-myc) antibodies. The results of one experiment, which were representative of two or three experiments, are shown. (B) CHO-T cells were transiently transfected with pcDNA3-myc-PH/PTB^WT or pcDNA3-myc-PH/PTB^S408A. After 24 h, the cells were starved for 16 h and treated either with 100 nM insulin for 30 min or with 25 nM calyculin A for 60 min or left alone (−). Cell extracts were resolved by SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-Myc antibodies. The results of one experiment, which were representative of two experiments, are shown.